The long-term goal of the research proposed here is to determine if distinct subtypes of voltage-gated Na channels coexist in the sarcolemma of skeletal muscle fibers and to ascertain the physiological role of these subtypes. The experiments proposed here will focus mainly on the endplate region of the muscle fibers, since preliminary data strongly suggests that a distinct Na channel subtype is localized in this region. The channel subtypes will be distinguished by differences in their kinetics and their susceptibility to modification of their tetrodotoxin (TTX) binding site by methylation with the drug trimethyloxonium (TMO) . These measurements will be performed using conventional loose and tight patch voltage clamping and a newly developed technique that combines loose patch voltage clamping with ionophoresis. This new technique will allow simultaneous measurement of both Na and acetylcholine (ACh)-induced current; thereby making it possible to distinguish subsynaptic Na channels from those in adjacent membrane, since subsynaptic Na currents will be correlated with a large ACh-induced response. Experiments will also be performed on denervated fibers to ascertain the relative effects of denervation on the separate channel subtypes since preliminary data suggest that channels near the endplate are more strongly affected by changes in synaptic input. Measurements will also be made of the return of normal TTX binding following in vivo TMO treatment to determine the change in channel turnover resulting from denervation. To ascertain if the same Na channel subtypes are expressed in slow twitch muscle fibers as in fast, measurements will be made in both fiber types. In conclusion, this research should provide significant new data concerning the number, distribution, and properties of Na channel subtypes in skeletal muscle fibers and the possible role that motor neurons may play in controlling the expression of these subtypes.